Molecular Cytogenetic Studies On Wheat-Thinopyrum Ponticum Derivatives And Development Of Specific Markers

Distant hybridization enriched the genetic diversity of wheat by introducing new exogenous materials,so as to achieve the purpose of directional genetic improvement.In this study,the derived progenies of wheat-Thinopyrum ponticum were screened and identified by morphology,cytology,disease resistance and molecular marker to select fantastic derivative materials with stability and comprehensive agronomic traits,and finally provide important intermediate materials and genetic resources for wheat breeding.What's more,the specific molecular markers of Th.ponticum were developed,which accelerated the detection of genetic material and laid a foundation for the excavating the excellent genes of Th.ponticum.The main results are as follows:(1)Preliminary karyotype analysis and the relationship wirh other species of Th.ponticum were analyzed.The relative length of 70 chromosomeswere calculated,including relative length coefficient,centromere index,arm ratio and other indexes by using the method of conventional cytology.The results showed that there were 35 pairs of chromosomes,of which,7 pairs were submetacentric chromosome(7sm),28 pairs were of metacentric chromosomes(28m),and 6 pairs were satellite chromosome(6sat).Therefore,it can be inferred that the karyotype formula of Th.ponticum was 2n=10x=70=56 m(8sat)+14 sm(4 sat),belonging to"2B"of Stebbins.At the same time,156 genomic specific markers(42 SSR and 114 EST)of Th.ponticum were identified from 879 pairs of SSR primers and EST primers.These molecular markers provided theoretical basis for the identification of exogenous genetic material in the derived progenies of Th.ponticum.The results of genetic diversity showed that 249 out of 897 primers amplified the same bands in Th.ponticum,Th.intermedium and Pseudoroegeria spicata,while 52 primers amplified the same bands in Th.ponticum,Psathyrostachys huashanica and Leymus mollis.This means that the relationship between Th.ponticum,Th.intermedium and Ps.spicata was closer than that of Ps.huashanica and L.mollis.(2)Nine stable partial diploids of Th.ponticum were identified from 228 derived progenies by classical cytogenetic method,named as CA51,CA52,CA53,CA61,CA62,CA63,CA64,CB14 and CB16.Cytological observations showed that almost all of the partial diploids contained 56 chromosomes and formed 28 bivalents during the first metaphase of meiosis,indicating that they were highly stable in cytology.The results of genomic in situ hybridization(GISH)showed that there were 40 chromosomes of common wheat and 16 chromosomes of Th.ponticum in CA51,CA52,CA53 and CA63.Four lines,CA61,CA62,CA64 and CB14 contained 42 chromosomes of common wheat and 14chromosomes of Th.ponticum.And CB16 had 12 chromosomes of Th.ponticum.Notably,a small translocated chromosome fragment from Th.ponticum was found in CA61 and CB16.Fluorescence in situ hybridization(FISH)was used to observe the variation of FISH karyotypes of nine partial diploids and the signal variation on chromosomes 1B,4B,6B and 5A of common wheat.The results of molecular markers showed that nine octoploids were different from those reported.In addition,eight octoploids showed strong resistance to stripe rust,powdery mildew and FHB at the adult stage,and nine of them had a great improvement on the grain quality,which provided high quality bridge materials for wheat distant hybridization.(3)Seven stable substitution lines were identified in this study,including 3 disomic substitution lines(CH10A5,CH114-B4,CH18122)and 4 double disomic substitution lines(CH18035,CH18050,CH18113,CH18130).The number of root tip chromosomes in each of them was 42,and the configuration of pollen mother cells(PMC)were 2n=21II.The field phenotype was similar to common wheat.Among them,CH10A5 was a 1J~S(1D)disomic subsitution line of wheat and Th.ponticum,which was highly resistant to stripe rust,moderately resistant to powdery mildew and FHB.CH114-B4 was a wheat-Th.ponticum 7J(7B)disomic subsitution line,showing high resistance to stripe rust and FHB,and moderate resistance to powdery mildew at adult stage.CH18122 was a 4J~S(4D)disomic subsitution line of wheat and Th.ponticum,showing high resistance to stripe rust,powdery mildew and FHB.The possible chromosome composition of CH18035 was 2n=38T.a+2T.p(1J~S)+2T.p(6J~S)(T.a and T.p refer to chromosomes of common wheat and Th.ponticum,respectively),indicating high susceptibility to stripe rust and powdery mildew at the adult stage.The possible chromosome composition of CH18050 was 2n=38T.a+2T.p(2J)+2T.p(7J),which was highly resistant to stripe rust and powdery mildew at adult stage.The possible chromosome composition of CH18113 was 2n=38T.a+2T.p(1J~S)+2T.p(6J),high resistance to stripe rust and high susceptibility to powdery mildew.The possible chromosome composition of CH18130 was 2n=38T.a+2T.p(2J)+2T.p(4J~S),showing high resistance to stripe rust and powdery mildew.The contents of crude protein,gluten,starch and stability time of the seven substitution lines were all higher than those of the common wheat parent 7182.And these substitution lines would be used as intermediate materials for wheat genetic improvement.(4)Based on SLAF sequencing,11,931,086,10,383,011 and 9,871,061 reads were obtained from CH10A5,7182 and Th.ponticum,respectively,with an average Q30 of90.34%and an average GC content of 46.55%.Furthermore,467,788,157,996,500,27SLAF tags were obtained,respectively.Based on the reference genome of CS,a total of564,364 polymorphism SNP loci were obtained by BWA,and 25,903 differential genes were annotated of Th.ponticum.The candidate genes related to the response to stress and grain were obtained by enrichment analysis in COG,GO and KEGG databases.Based on the unreferenced genome,1,096 pairs of primers were selected from 18,971 unmapped SLAFs of Th.ponticum,and 859 specific markers were obtained by PCR amplification and electrophoresis.At the same time,primers were designed from 957 sequences on the 1J~Schromosome of CH10A5,which were 95%similar to Th.ponticum.And 507 pairs of STS primers amplified same specific bands on CH10A5 and Th.ponticum.In order to analyze the polymorphism and practicability of these specific primers,some wheat-related species were evaluated.The results showed that some of markers had polymorphism in Th.ponticum(49),Th.bessarabicum(38),Th.elongatum(45),Pseudoroegeria spicata(89),Th.intermedium(57);in addition,other related species of wheat were amplified specific bands,including L.mollis,Ps.spicata,rye,Ae.geniculata,T.urartu and Ae.tauschii,and their specific markers were 70,51,67,54,17,and 43,respectively.Based on the number of specific molecular markers in wheat and its related species,the distance of genetic relationship between wheat and its related species was predicted.The chromosomal specific markers were developed in this study,including 21 specific markers on the St genome,21 specific markers on the E genome,15 specific markers on the E~e genome and 6specific markers on the E~b genome,which provided a theoretical basis for the subsequent detection of genetic materials and chromosome rearrangement of Th.ponticum.